Not Applicable
The field of this invention is the area of molecular biology, in particular, as related to tissue specific promoters of plants, and more particularly, to promoters which selectively direct expression of genetic information in the developing seedpod and certain other tissues of a legume or other plant.
There is a longfelt need in the art for expression control sequences which mediate tissue specific and/or developmentally regulated expression of nucleotide sequences of interest in plants. The present invention fills this need.
The present invention provides transcriptional regulatory sequences which mediate tissue specific and developmental stage specific expression of nucleotide sequences placed under the control of those transcriptional regulatory sequences, The tissue specificity of the transcription regulatory sequences of the present invention is such that expression occurs preferentially in the developing seedpod of a plant, especially of a leguminous plant, for example, soybean, green bean, or pea. The Msg transcription regulatory sequences also direct expression in guard cells of stem, in roots, shoots, node cells and flowers. The sequences of the present invention function in monocots or dicots. As specifically exemplified herein, the developing seedpod-specific promoter of the gene termed Msg herein, is from the genome of Glycine max. The transcriptional regulatory sequences of the present invention can include sequences which direct the initiation of transcription, for example the entire untranslated region, e.g., nucleotides xe2x88x922260 to xe2x88x921 of Table 1, relative to the translation start site, or xe2x88x922260 to about xe2x88x92650 in the absence of other promoter sequences, or the sequences can be positioned upstream of and in the same orientation as a truncated promoter region (a truncated promoter region desirably includes a TATA region, and optionally at least one CAAT element). Chimeric genes of the present invention include transcription regulatory sequences which direct tissue-specific and developmental stage specific expression of downstream nucleotide sequences, primarily in developing and maturing seedpods, but not in developing seeds. The sequence downstream of the transcription regulatory element of the present invention can be any of a number of sequences including but not limited to a pathogenesis-related protein, a plant defense protein, lectins with specific binding activities, an antibacterial protein of a plant or animal, an insect-resistance gene such as a naturally occurring or a Bacillus thuringiensis insecticidal protein expressible in plants or a chitinase or a fungal resistance protein. Alternatively, the sequence to be expressed in the specifically regulated manner of the present invention can be a regulatory nucleic acid sequence whose expression is desired in the seedpod or guard cells in stem, roots, shoots, node cells and flowers, and the 3xe2x80x2UTR directs expression in the filaments of stamens.
The transcription regulatory sequences of the present invention can be isolated, using the specifically exemplified sequences as hybridization probes or to generate primers for polymerase chain reaction (PCR) amplification, from a legume, including but not limited to, soybean, green bean, other beans, pea, alfalfa, clover, among others, such as those plants which have dehiscent (dry) fruit including the Brassicaceae (canola, cabbage, mustard, broccoli, etc.), some members of the Solanaceae (e.g., tobacco), the Linaceae (e.g., flax and linseed), the Papaveraceae, and the horticultural species of the Ranunculaceae. Certain members of the Curcurbitaceae family, such as muskmelons and cucumbers, are also improved in pathogen resistance and/or drought resistance by expression of the exemplified Msg gene or by introduction of another pathogen resistance protein expressed under the control of the Msg transcription regulatory sequences. The nucleotide sequence of a specifically exemplified soybean pod-specific transcription regulatory region (of the Msg gene), including full promoter, is given in Table 1 herein. Shorter sequences can be derived from the exemplified sequence, where the shorter sequences are effective to regulate the transcriptional expression of a nucleotide sequence under their control. Specifically exemplified shorter sequences which retain the ability to regulate transcriptional expression of a downstream nucleotide sequence include those sequences of Table 1 and having limits as set forth in FIG. 6. It is understood that sequences in the region of about xe2x88x922260 to about xe2x88x92650, or about xe2x88x922274 to xe2x88x921, or about xe2x88x922274 to about xe2x88x92650, relative to the translation site, direct the initiation of transcription in the desired tissue-specific and/or developmentally regulated manner.
The present invention further provides plant transformation vectors which comprise the transcription regulatory sequences of the present invention together with operably linked downstream sequences to be expressed in a plant in a tissue specific and developmental pattern specific fashion. The vectors can be introduced into plant cells or tissue by any methods known to art, including but not limited to, Agrobacterium-mediated transformation, electroporation, microinjection and microprojectile bombardment. Regeneration of plants from transformed cells and tissue is well known to the art.
Also within the scope of the present invention are plant cells, plant tissue, transgenic plants, pod cells, seeds, etc., which contain a heterologous nucleotide sequence expressed under the regulatory control of the pod specific transcription regulatory sequences described herein.
Additional objects of the present invention include the production of transgenic plant cells, tissue and plants in which a nucleotide sequence of interest is operably linked to transcription control sequences which mediate expression of the sequence of interest in a tissue specific fashion, preferentially in the seedpod (but not in the seed) of such a transgenic plant. Additional sites of expression of the Msg-regulated transcript include, but are not limited to, guard cells of stem, cells in nodes, shoots, roots and flowers, but not seeds.
The method of the present invention comprises the step of introducing a recombinant DNA construct comprising an Msg-regulated nucleotide sequence into a plant cell or into plant tissue, such that the construct becomes stably incorporated within the genome of said plant cell. In the present context, the construct can simply be a sequence operably linked to the transcription regulatory sequences of the present invention, or the construct can further include a selective marker which readily allows the skilled artisan to determine incorporation of the construct, and the construct can comprise vector sequences which allow amplification of the construct in a host cell other than a plant cell (e.g., the vector sequences can be a plasmid sequences). Transgenic plants are regenerated from the stably transformed plant cells or tissue according to art-known techniques.